Increased expression of cyclooxygenase-2 in the renal cortex of human prorenin receptor gene-transgenic rats
2006; Elsevier BV; Volume: 70; Issue: 4 Linguagem: Inglês
10.1038/sj.ki.5001627
ISSN1523-1755
AutoresYuki Kaneshiro, Atsuhiro Ichihara, Tomoko Takemitsu, Mariyo Sakoda, Fumihiko Suzuki, Tsutomu Nakagawa, Matsuhiko Hayashi, Tadashi Inagami,
Tópico(s)Renin-Angiotensin System Studies
ResumoIncreased macula densa cyclooxygenase-2 (COX-2) is observed in diabetic rats and may contribute to hyperfiltration states. However, the signals mediating increased COX-2 expression in diabetic rats remain undetermined. We recently found that non-proteolytic activation of prorenin by site-specific binding proteins, such as prorenin receptor, plays a pivotal role in the development of diabetic nephropathy. The present study was designed to determine the contribution of prorenin receptor to renal cortical COX-2 expression. The COX-2 mRNA and protein levels of six 4-week-old male wild-type rats and six human prorenin receptor gene-transgenic (hProRenRcTg) rats were measured by real-time polymerase chain reaction methods, Western blotting, and immunohistochemistry, and compared. There were no differences between the two groups in arterial pressure measured by telemetry, urinary sodium excretion, or renal levels of rat prorenin receptor mRNA. The renal cortical COX-2 mRNA levels of the hProRenRcTg rats were significantly higher than those of the wild-type rats, and the renal cortical COX-2 protein levels were also higher in hProRenRcTg rats than in the wild-type rats. Immunohistochemical analysis revealed that COX-2 immunostaining was predominantly present in the macula densa cells, and significantly more COX-2-positive cells were present in the hProRenRcTg rats than in the wild-type rats. In addition, COX-2 inhibition with NS398 significantly decreased renal cortical blood flow in the hProRenRcTg rats but not in the wild-type rats. These results strongly suggest that human prorenin receptor directly or indirectly contributes to the regulation of renal cortical COX-2 expression. Increased macula densa cyclooxygenase-2 (COX-2) is observed in diabetic rats and may contribute to hyperfiltration states. However, the signals mediating increased COX-2 expression in diabetic rats remain undetermined. We recently found that non-proteolytic activation of prorenin by site-specific binding proteins, such as prorenin receptor, plays a pivotal role in the development of diabetic nephropathy. The present study was designed to determine the contribution of prorenin receptor to renal cortical COX-2 expression. The COX-2 mRNA and protein levels of six 4-week-old male wild-type rats and six human prorenin receptor gene-transgenic (hProRenRcTg) rats were measured by real-time polymerase chain reaction methods, Western blotting, and immunohistochemistry, and compared. There were no differences between the two groups in arterial pressure measured by telemetry, urinary sodium excretion, or renal levels of rat prorenin receptor mRNA. The renal cortical COX-2 mRNA levels of the hProRenRcTg rats were significantly higher than those of the wild-type rats, and the renal cortical COX-2 protein levels were also higher in hProRenRcTg rats than in the wild-type rats. Immunohistochemical analysis revealed that COX-2 immunostaining was predominantly present in the macula densa cells, and significantly more COX-2-positive cells were present in the hProRenRcTg rats than in the wild-type rats. In addition, COX-2 inhibition with NS398 significantly decreased renal cortical blood flow in the hProRenRcTg rats but not in the wild-type rats. These results strongly suggest that human prorenin receptor directly or indirectly contributes to the regulation of renal cortical COX-2 expression. Cyclooxygenase-2 (COX-2) is constitutively present in the macula densa cells and in medullary interstitial cells of the kidney.1.Kujubu D.A. Fletcher B.S. Varnum B.C. et al.TIS10, a phorbol ester tumor promoter-inducible mRNA from swiss3T3 cells, encodes a novel prlstaglandin synthase/cyclooxygenase homologue.J Biol Chem. 1991; 266: 12866-12872Abstract Full Text PDF PubMed Google Scholar,2.Xie W. Chipman J.G. Robertson D.L. et al.Expression of a mitogen-responsive gene encoding prostaglandin systhase is regulated by mRNA splicing.Proc Natl Aca Sci USA. 1991; 88: 2692-2696Crossref PubMed Scopus (1643) Google Scholar COX-2 activity in the macula densa is stimulated by high renal perfusion pressure,3.Ichihara A. Imig J.D. Navar L.G. Cyclooxygenase-2 modulates afferent arteriolar responses to increases in pressure.Hypertension. 1999; 34: 843-847Crossref PubMed Google Scholar and the stimulated macula densa COX-2 catalyzes the synthesis of vasodilator prostaglandins counteracting the tubuloglomerular-feedback-mediated afferent arteriolar constriction.4.Ichihara A. Imig J.D. Inscho E.W. Navar L.G. Cyclooxygenase-2 participates in tubular flow-dependent afferent arteriolar tone: interaction with neuronal NOS.Am J Physiol. 1998; 275: F605-F612PubMed Google Scholar Thus, increased activity of macula densa COX-2 can elicit a decrease in afferent arteriolar resistance, which leads to elevation of intraglomerular pressure.5.Peterdi J.P. Komlosi P. Fuson A.L. et al.Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells.J Clin Invest. 2003; 112: 76-82Crossref PubMed Scopus (121) Google Scholar Recent studies have shown increased expression of macula densa COX-2 in the kidneys of diabetic humans6.Khan K.N.M. Burke A. Stanfield K.M. et al.Expression of cyclooxygenase-2 in the macula densa of human kidney in hypertension, congestive heart failure, and diabetic nephropathy.Renal Failure. 2001; 23: 321-330Crossref PubMed Scopus (58) Google Scholar and rats.7.Komers R. Lindsly J.N. Oyama T.T. et al.Immunohistochemical and functional correlations of renal cyclooxygenase-2 in experimental diabetes.J Clin Invest. 2001; 107: 889-898Crossref PubMed Scopus (174) Google Scholar Because dilated afferent arterioles and enlarged glomeruli are observed in the early stage of diabetic nephropathy, the increased expression of macula densa COX-2 may account for the hyperfiltration state of the glomeruli owing to afferent arteriolar dilation in diabetic kidneys. As glomerular hyperfiltration is well known to play an important role in the development of diabetic nephropathy, macula densa COX-2 may contribute to the development of nephropathy in diabetic kidneys. However, the mechanism of the upregulation of macula densa COX-2 expression in diabetic kidneys remained unknown. We recently found that site-specific prorenin-binding proteins, such as prorenin receptor,8.Nguyen G. Delarue F. Burckle C. et al.Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.J Clin Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1136) Google Scholar contribute to the development of diabetic nephropathy,9.Ichihara A. Hayashi M. Kaneshiro Y. et al.Inhibition of diabetic nephropathy by a decoy peptide corresponding to the 'handle' region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (383) Google Scholar and we therefore hypothesized that the activation of prorenin receptor also contributes to the mechanism of the increased expression of COX-2 in the macula densa in diabetic kidneys. To test this hypothesis, in the present study we assessed the expression and function of renal cortical COX-2 in rats expressing a high level of human prorenin receptor (hProRenRc). Figure 1 shows that hProRenRc transgene was distributed to various tissues including brain, heart, kidney, liver, aorta, femoral artery, testis, lung, spleen, pancreas, adipose tissue, adrenal gland, ovary, and uterus. In the kidneys of hProRenRc-transgenic (Tg) rats, the majority of hProRenRc transgene was present in tubular cells, and a few were observed in the glomerular epithelium. As shown in Table 1, there were no significant differences in mean atrial pressure (MAP), urinary sodium excretion, plasma renin activity, plasma angiotensin II level, kidney angiotensin II level, renal plasma flow, and rat prorenin receptor (rProRenRc) mRNA levels between the Tg and wild-type rats. The microhematocrit of blood samples from wild-type and Tg rats was also similar and remained constant throughout the experiment. Urinary prostaglandin E2 was significantly higher in Tg rats than in wild-type rats. The mRNA expression of hProRenRc was detected only in Tg rats.Table 1Characterization of hProRenRc gene-transgenic (Tg) ratsWildTgP-valueNumber66MAP (mm Hg)88±288±10.54UNaV (mEq/day)3.67±0.193.58±0.300.84Urinary PGE2 (ng/h)20.3±1.833.7±2.0<0.001Microhematocrit (%)54.3±1.752.3±0.90.30PRA (ng/ml/h)3.3±0.9 (n=4)2.5±0.6 (n=19)0.43Plasma Ang II (fmol/l)48.6±7.2 (n=14)37.3±6.5 (n=19)0.26Kidney Ang II (fmol/g)53.7±7.840.1±9.00.30Renal plasma flow (ml/min/g)5.7±0.65.4±0.50.81Kidney rProRenRc mRNA (ratio to rat GAPDH mRNA)6.9±0.56.8±0.40.88Data are shown as means±s.d.Ang II, angiotensin II; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, mean arterial pressure; PGE2, prostaglandin E2; PRA, plasma renin activity; rProRenRc, rat prorenin receptor; UNaV, urinary sodium excretion. Open table in a new tab Data are shown as means±s.d. Ang II, angiotensin II; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, mean arterial pressure; PGE2, prostaglandin E2; PRA, plasma renin activity; rProRenRc, rat prorenin receptor; UNaV, urinary sodium excretion. Figure 2a shows the COX-2 mRNA levels in the renal cortex of the Tg and wild-type rats determined quantitatively by a real-time polymerase chain reaction method. Renal cortical COX-2 mRNA level in the Tg rats was 5.33±0.70 (ratio to glyceraldehyde-3-phosphate dehydrogenase mRNA) and was significantly higher than in the wild-type rats (1.28±0.66 (ratio to glyceraldehyde-3-phosphate dehydrogenase mRNA)). Figure 2b shows the COX-2 protein levels in the renal cortex of the Tg and wild-type rats determined by Western blot analysis. Renal cortical COX-2 protein levels (1.06±0.26 (ratio to α-tubulin)) were also higher in Tg rats than in the wild-type rats (2.18±0.17 (ratio to α-tubulin)). Figure 3 shows the results of immunostaining with anti-rat COX-2 antibody in the renal cortex of the Tg and wild-type rats. Strong COX-2 immunostaining was observed in the macula densa cells of the hProRenRcT rats, but weak staining in the renal cortex of the wild-type rats. Both the Tg and wild-type rats had a similar value of renal blood flow because they had similar values of renal plasma flow, as shown in Table 1, and microhematocrit throughout the experiment. Figure 4 shows the effects of the COX-2 inhibitor NS398 on renal blood flow assessed by an electromagnetic flow probe and renal cortical blood flow in the Tg and wild-type rats. There were no significantly changes in MAP in either group during COX-2 inhibition with NS398. Under anesthesia, MAP at the beginning and end of the study period measured 122±3 and 117±6 mm Hg, respectively, in the wild-type rats, and 118±2 and 120±3 mm Hg, respectively, in the Tg rats. NS398 did not significantly alter renal blood flow or renal cortical blood flow in the wild-type rats; however, the 1 and 10 ng/g bw (gram body weight) doses of NS398 significantly reduced the renal blood flow of the Tg rats from 2.91±0.23 to 2.39±0.19 and 2.03±0.41 ml/min, respectively, and decreased the renal cortical blood flow of the Tg rats by 27.2±3.6 and 42.3±7.9%, respectively. The decreases in renal blood flow and renal cortical blood flow in the Tg rats compared to the wild-type rats were significant. Figure 5 shows the phosphorylated and total extracellular signal-regulated kinase (ERK) levels in the renal cortex of the Tg and wild-type rats determined by Western blot analysis. The ratio of phospho-ERK level to total ERK level in the renal cortex was significantly higher in Tg rats (4.7±0.5) than in the wild-type rats (1.8±0.4). Transgenic expression of hProRenRc in rats did not affect MAP, urinary sodium excretion, or rProRenRc levels. However, macula densa COX-2 expression was significantly higher in the Tg rats than in the wild-type rats, and the increased COX-2 expression significantly contributed to the regulation of renal cortical blood flow. These results suggest that hProRenRc expression causes an increase in expression and function of macula densa COX-2 independently of blood pressure or urinary sodium excretion. When prorenin binds to the prorenin receptor, two important signals are thought to occur. One signal is the non-proteolytic activation of prorenin: prorenin undergoes a conformational change that includes exposure of its enzymatically active site, and the activated prorenin stimulates the renin–angiotensin system (RAS).9.Ichihara A. Hayashi M. Kaneshiro Y. et al.Inhibition of diabetic nephropathy by a decoy peptide corresponding to the 'handle' region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (383) Google Scholar The other signal is an increase in mitogen-activated protein kinase (MAPK)-dependent intracellular transduction that is independent of the RAS.8.Nguyen G. Delarue F. Burckle C. et al.Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin.J Clin Invest. 2002; 109: 1417-1427Crossref PubMed Scopus (1136) Google Scholar,10.Schefe J.H. Kaiser H.F. Jost A. Unger T. Signal transduction of the renin/prorenin receptor.J Hypertens. 2005; 123: S354-S355Google Scholar Thus, enhancement of macula densa COX-2 expression by hProRenRc is thought to be mediated through both a RAS-dependent mechanism and a RAS-independent mechanism. A recent study showed that angiotensin II stimulates renal COX-2 mRNA expression in 5/6 renal-ablated rats.11.Hernandez J. Astudillo H. Escalante B. Angiotenin II stimulates cyclooxygenase-2 mRNA expression in renal tissue from rats with kidney failure.Am J Physiol. 2002; 282: F592-F598PubMed Google Scholar Human ProRenRc may stimulate renal COX-2 mRNA expression through an increase in angiotensin II levels owing to RAS activation. In the present study, however, plasma angiotensin II levels were similar in the Tg and wild-type rats. In addition, Cheng et al.12.Cheng H.F. Wang J.L. Zhang M.Z. et al.Angiotensin II attenuates renal cortical cyclooxygenase-2 expression.J Clin Invest. 1999; 103: 953-961Crossref PubMed Scopus (189) Google Scholar clearly demonstrated that angiotensin II attenuates renal COX-2 expression localized to the cortical thick ascending limb of Henle's loop cells in the region of the macula densa in the rats. It is therefore unlikely that the RAS activation contributes to the increase in macula densa COX-2 expression by hProRenRc. Low-chloride medium stimulates COX-2 expression in primary cultured cortical thick ascending limb of Henle's loop cells13.Cheng H.F. Wang J.L. Zhang M.Z. et al.Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride.J Clin Invest. 2000; 106: 681-688Crossref PubMed Scopus (111) Google Scholar and a macula densa cell line14.Yang T. Park J.M. Arend L. et al.Low chloride stimulation of prostaglandin E2 release and cyclooxygenase-2 expression in a mouse macula densa cell line.J Biol Chem. 2000; 275: 37922-37929Crossref PubMed Scopus (127) Google Scholar through phosphorylation of the three major MAPK pathways, p38, Jun N-terminal kinase, and ERK, suggesting that MAPK pathways play an important role in the expression of COX-2 localized to the region of macula densa. In the present study, the ratio of phospho-ERK to total ERK was significantly increased in the renal cortex from Tg rats compared to wild-type rats, suggesting that hProRenRc may stimulate the ERK activation. Therefore, phosphorylation of the MAPK pathways by hProRenRc can mediate the upregulation of macula densa COX-2 expression observed in the present study. Further studies are needed to clarify the mechanism of the upregulation of macula densa COX-2 by hProRenRc. As COX-2 mediates the kidney prorenin synthesis induced by a low-salt diet,15.Harding P. Sigmon D.H. Alfie M.E. et al.Cyclooxygenase-2 mediates increased renal renin content induced by low-sodium diet.Hypertension. 1997; 29: 297-302Crossref PubMed Google Scholar increased levels and/or activity of macula densa COX-2 can activate RAS and lead to blood pressure elevation and a decreased urinary sodium excretion. In the present study, however, both the blood pressure and urinary sodium excretion of the Tg rats were similar to the values in the wild-type rats, and the plasma renin activity, plasma angiotensin II levels, and kidney angiotensin II levels of 4-week-old Tg rats were similar to those of 4-week-old wild-type rats. Thus, it appeared that the plasma or kidney RAS of the Tg rats is not activated at 4 weeks of age. Nevertheless, renal cortical ERK was activated in the Tg rats compared to the wild-type rats. Because of the high homology between the amino-acid sequences of rProRenRc and hProRenRc, rat prorenin is able to bind to hProRenRc. Our preliminary study using COS-7 cells that expressed hProRenRc showed that rat prorenin is capable of binding to hProRenRc but does not enzymatically activate prorenin. The maximum binding of hProRenRc to rat prorenin was 70% of the maximum binding of hProRenRc to human prorenin, and the Kd value of rat prorenin binding to hProRenRc was 3 nmol/l and approximately two-fold higher than the Kd of human prorenin binding to hProRenRc. In the presence of rat prorenin, however, sheep angiotensinogen was not converted to angiotensin I in the medium of COS-7 cells that expressed hProRenRc. Therefore, transgenic expression of hProRenRc was capable of stimulating the RAS-independent intracellular signals, but could not activate the RAS, because of competitive inhibition of rProRenRc binding to rat prorenin by hProRenRc. This may account for no activation of the RAS despite increased direct actions of prorenin in the Tg rats. In the present study, the clearance method and electromagnetic flow probe measurements confirmed a similar baseline renal plasma flow in wild-type and Tg rats despite enhanced COX-2 expression in the renal cortex of Tg rats. These results suggest that some compensative mechanisms worked to keep the renal plasma flow constant against increased COX-2 expression. Therefore, COX-2 inhibition with NS398 caused a significant decrease in renal cortical blood flow only in Tg rats, which had an increased level of macula densa COX-2. In conclusion, transgenic expression of hProRenRc resulted in stimulation of the expression and activity of macula densa COX-2. Thus, hProRenRc may influence the macula densa-dependent renal microcirculation by regulating COX-2 expression. These experiments were performed in accordance with the guidelines and practices established by the Keio University Animal Care and Use Committee. hProRenRc (GenBank accession number AF291814; nt 73–1301) plasmid vector, designated pCAGGS-hProRenRc, was used. The details of its use, including its construction, have been described previously.16.Niwa H. Yamamura K. Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4424) Google Scholar Briefly, we constructed an expression vector by introducing a CAG promoter that comprises an AG promoter with a cytomegalovirus immediate early gene enhancer and rabbit β-globin gene terminator sequences including a polyadenylation signal. This vector, designated pCAGGS-hProRenRc, ubiquitously exhibited high-level production of hProRenRc in various organs. Transgenic rats were generated by pronuclear microinjection of fertilized Crj:Wistar rat eggs and reimplantation into pseudopregnant Crj:Wistar foster mothers. Six foster mothers carried 31 pups to term, three of which carried the transgene, as demonstrated by PCR analysis of DNA obtained from tail blood. Two of these founders transmitted the transgene to their progeny, and the transgenic lines hProRenRc no. 21 line and no. 29 line were established. Transgene of hProRenRc was ubiquitously observed in the brain, heart, kidney, liver, aorta, arteries, testis, lung spleen, pancreas, adipose tissue, adrenal gland, ovary, and uterus, as shown in Figure 1. The no. 21 line was used for the present study. Arterial pressure was determined under the unrestricted and untethered condition in 4-week-old rats using a telemetry device inserted into the abdominal aorta, and the 24-h urine was collected in metabolic cage. To determine p-aminohippurate sodium clearance as a renal plasma flow, renal clearance study was performed under anesthesia in the rats continuously infused with isotonic saline solution containing 1% albumin and 1.5% p-aminohippurate sodium (Merck & Co., Inc., Whitehouse Station, NJ, USA) at a rate of 20 μl/min, as described previously.17.Tada Y. Ichihara A. Koura Y. et al.Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.Kidney Int. 2004; 66: 1966-1976Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Two groups of rats (n=6 each) were used in this series of experiments: a wild-type group born of the hProRenRc line but to which the hProRenRc gene had not been transmitted (wild), and a group of hProRenRc gene-transgenic (Tg) rats. We decapitated six rats of each group to obtain the blood and kidneys. Immediately after decapitation, a 3-ml blood specimen was collected into a tube containing 30 μl of ethylenediaminetetraacetic acid (500 mM), 15 μl of enalaprilat (1 mM), and 30 μl of o-phenanthroline (24.8 mg/ml) and pepstatin (0.2 mM), and plasma samples were obtained by centrifugation. Plasma renin activity was determined with a radioimmunoassay coated-bead kit (Dinabott Radioisotope Institute, Tokyo, Japan). For the determination of kidney angiotensin II content, the removed kidney was weighed, placed in ice-cold methanol (10% wt/vol), homogenized with a chilled glass homogenizer, and centrifuged. The supernatant was then dried and reconstituted in 4 ml of 50 mM sodium phosphate buffer containing 1% albumin. Plasma and reconstituted samples from the kidneys were extracted with a Bond-Elut column (Analytichem, Harbor City, CA, USA), and the eluents were evaporated to dryness and reconstituted in angiotensin peptide assay diluent. The angiotensin II content was quantitatively determined by radioimmunoassay using rabbit anti-angiotensin II antiserum (Arnel, New York, NY, USA). The urine samples were collected in a metabolic cage, and the urinary concentration of prostaglandin E2 was determined by radioimmunoassay using a [125I] radioimmunoassay kit (PerkinElmer Life Sciences Inc., Boston, MA, USA) after the extraction by conventional methods and subsequent purification on Bond-Elut Si columns (Analytichem International, Harbor City, CA, USA). As described previously,9.Ichihara A. Hayashi M. Kaneshiro Y. et al.Inhibition of diabetic nephropathy by a decoy peptide corresponding to the 'handle' region for nonproteolytic activation of prorenin.J Clin Invest. 2004; 114: 1128-1135Crossref PubMed Scopus (383) Google Scholar total RNA from part of the renal cortex removed from each animal was extracted with an RNeasy Mini Kit (QIAGEN KK, Tokyo, Japan), and real-time quantitative reverse transcriptase-PCR (RT-PCR) was performed by using the TaqMan One-Step RT-PCR Master Mix Reagents Kit with an ABI Prism 7700 HT Detection System (Applied Biosystems Inc., Foster City, CA, USA) and probes and primers for rat genes encoding the following: COX-2 (forward, 5′-TGTTCGCATTCTTTGCCCA-3′; reverse, 5′-TAAGTCCACTCCATGGCCCA-3′; probe, 5′-FAM-TCAGAAGCGAGGACCTGGGTTCACC-TAMRA-3′); rProRenRc (forward, 5′-CATTCGACACATCCCTGGTG-3′; reverse, 5′-AAGGTTGTAGGGACTTTGGGTG-3′; probe, 5′-FAM-AAGTCAAGGACCATCCTTGAGACGAAACAA-TAMRA-3′); hProRenRc (forward, 5′-AGATGACATGTACAGTCTTTATGGTGG-3′; reverse, 5′-TGCTGGGTTCTTCGCTTGT-3′; probe, 5′-FAM-TTTGACACCTCCCTCATTAGGAAGACAAGGACT-TAMRA-3′); and glyceraldehyde-3-phosphate dehydrogenase (forward, 5′-TGACAACTCCCTCAAGATTGTCA-3′; reverse, 5′-GGCATGGACTGTGGTCATGA-3′; probe, 5′-FAM-TGCATCCTGCACCACCAACTGCTTAG-TAMRA-3′). Renal localization of the transgene was assessed by in situ hybridization, as described previously.18.Burckle C.A. Jan Danser A.H. Muller D.N. et al.Elevated blood pressure and heart rate in human renin receptor Transgenic rats.Hypertension. 2006; 47: 1-5Crossref PubMed Scopus (186) Google Scholar Briefly, digoxigenin-labeled cRNA antisense and sense probes were synthesized using a 600-bp fragment of the AF291814 as template. Western blot analyses were performed as reported previously.17.Tada Y. Ichihara A. Koura Y. et al.Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.Kidney Int. 2004; 66: 1966-1976Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Briefly, the renal cortex was lysed in Tris (25 mmol/l) containing NaCl (150 mmol/l), glycerol (5%), and protease inhibitor (1 tablet/25 ml buffer), and after centrifugation at 10 000 g for 5 min followed by centrifugation at 24 000 g for 30 min, the supernatant was collected and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes, and after blocking the blots overnight with phosphate-buffered saline containing 5% bovine albumin and 0.5% Tween 20, they were incubated for 16 h with mouse monoclonal anti-rat COX-2 antibody (1:1000 dilution; Exalpha Biologicals, Watertown, MA, USA), mouse monoclonal anti-phospho-44/42 MAPK (E10) antibody that crossreacts with rat phosphorylated ERK1 and 2 (1:800 dilution; Cell Signaling Technology, Beverly, MA, USA), or mouse monoclonal anti-α-tubulin antibody (1:500 dilution; EMD Biosciences, La Jolla, CA, USA). Immunoreactivity was determined by horseradish peroxidase-conjugated donkey anti-mouse antibody and an enhanced chemiluminescence reaction, and the quantitative analyses were performed with Image 1D (Pharmacia, Peapack, NJ, USA). Immunohistochemical staining for COX-2 was performed as reported previously.17.Tada Y. Ichihara A. Koura Y. et al.Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.Kidney Int. 2004; 66: 1966-1976Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Briefly, part of the kidney was fixed in 4% paraformaldehyde phosphate buffer solution before paraffin embedding, and deparaffinized sections were pretreated with proteinase K. The sections were then boiled in citrate buffer with microwaves to unmask antigenic sites, and endogenous biotin was blocked with a biotin-blocking system (X0590; DAKO Corp., Kyoto, Japan). Next, the sections were immersed in 0.3% H2O2 in methanol to inhibit endogenous peroxidase and then precoated with 1% nonfat milk in phosphate-buffered saline to block nonspecific binding. Polyclonal rabbit anti-COX-2 antibody (160116, Cayman Chemical, Ann Arbor, MI, USA) (1:200–500 dilution) was used as the primary antibodies, and a biotinylated polyclonal goat anti-rabbit antibody (1:500 dilution) was applied as the second antibody. Immunoreaction was performed with a Vectastain ABC-Elite kit (Vector Laboratories, Burlingame, CA, USA) and visualized with 0.02% 3′,3′-diaminobenzidine tetrahydrochloride as a substrate, and then lightly counterstaining with hematoxylin. The effect of the COX-2 inhibitor NS398 (Cayman Chemical, Ann Arbor, MI, USA) on renal cortical blood flow was assessed as reported previously.17.Tada Y. Ichihara A. Koura Y. et al.Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.Kidney Int. 2004; 66: 1966-1976Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Briefly, rats were anesthetized with intraperitoneal pentobarbital sodium (50 mg/kg). The right femoral artery was cannulated, and the cannula was connected to a computer system (MacLab/8s, AD Instruments, Nagoya, Japan) via a pressure transducer to monitor arterial pressure. The right carotid artery was also cannulated to provide separate access for intra-arterial bolus doses of NS398. The left kidney was exposed by a flank incision and placed in a Lucite cup to stabilize it. A needle flow probe (500-μm diameter) connected to a laser-Doppler flowmeter (FloC1-Twin; Omegawave, Tokyo, Japan) was inserted into the kidney mass to a depth of 1 mm to position its tip in the superficial cortex, and used to measure relative changes in renal cortical blood flow. In addition, a small-diameter, noncannulating, and factory-precalibrated electromagnetic flow probe (1RB2949; Transonic Systems Inc., Ithaca, NY, USA), connected to a transit time flowmeter module (D-79232; Transonic Systems Inc.), was vertically fitted around the left renal artery to measure renal blood flow continuously. The rats were given intra-arterial bolus injections (100 μl) of increasing doses of NS398 (0, 1, and 10 ng/g) at 10-min intervals. Within-group statistical comparisons were made by one-way analysis of variance for repeated measures followed by the Neuman–Keuls post hoc test. Differences between two groups were evaluated by two-way analysis of variance for repeated measures combined with the Newman–Keuls post hoc test. A P-value of <0.05 was considered significant. Data are reported as means±s.e.m. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan (16613002, 16790474, 17390249, and 41503340) and research Grant HL58205 from the United States National Institutes of Health.
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